Definition of a consensus DNA‐binding site for the Escherichia coli pleiotropic regulatory protein, FruR

The FruR regulator of Escherichia coli controls the initiation of transcription of several operons encoding a variety of proteins involved in carbon and energy metabolism. The sequence determinants of the FruR‐binding site were analysed by using 6× His‐tagged FruR and a series of double‐stranded randomized oligonucleotides. FruR consensus binding sites were selected and characterized by several consecutive rounds of the polymerase chain reaction‐assisted binding‐site selection method (BSS) using nitrocellulose‐immobilized DNA‐binding protein. FruR was demonstrated to require, for binding, an 8 bp left half‐site motif and a 3 bp conserved right half‐site with the following sequence: 5′‐GNNGAATC/GNT‐3′. In this sequence, the left half‐site AATC/ consensus tetranucleotide is a typical motif of the DNA‐binding site of the regulators of the GalR–LacI family. On the other hand, the high degree of degeneracy found in the right half‐site of this palindrome‐like structure indicated that FruR, which is a tetramer in solution, interacts asymmetrically with the two half‐sites of its operator. However, potentially FruR‐target sites showing a high degree of symmetry were detected in 13 genes/operons. Among these, we have focused our interest on the pfkA gene, encoding phosphofructokinase‐1, which is negatively regulated by FruR.

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